Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K⁺ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H⁺ load on the cytosol and promoted Ca²⁺ influx to elevate cytosolic pH (pH_i) and free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), in turn regulating the K⁺ channels. We have confirmed these predictions, measuring pH_i and [Ca²⁺]_i in vivo, and report that experimental manipulation of pH_i and [Ca²⁺]_i is sufficient to recover K⁺ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K⁺ channels. Additionally, these findings underscore the importance of H⁺-coupled anion transport for pH_i homeostasis.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K⁺ channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K⁺ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K⁺ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

The plasma membrane proton (H⁺)-ATPases of plants generate steep electrochemical gradients and activate osmotic solute uptake. H⁺-ATPase-mediated proton pumping orchestrates cellular homeostasis and is a prerequisite for plastic cell expansion and plant growth. All evidence suggests that the population of H⁺-ATPase proteins at the plasma membrane reflects a balance of their roles in exocytosis, endocytosis, and recycling. Auxin governs both traffic and activation of the plasma membrane H⁺-ATPase proteins already present at the membrane. As in other eukaryotes, in plants, SNARE-mediated membrane traffic influences the density of several proteins at the plasma membrane. Even so, H⁺-ATPase traffic, its relationship with SNAREs, and its regulation by auxin have remained enigmatic. Here, we identify the Arabidopsis (Arabidopsis thaliana) Qa-SNARE SYP132 (Syntaxin of Plants132) as a key factor in H⁺-ATPase traffic and demonstrate its association with endocytosis. SYP132 is a low-abundant, secretory SNARE that primarily localizes to the plasma membrane. We find that SYP132 expression is tightly regulated by auxin and that augmented SYP132 expression reduces the amount of H⁺-ATPase proteins at the plasma membrane. The physiological consequences of SYP132 overexpression include reduced apoplast acidification and suppressed vegetative growth. Thus, SYP132 plays unexpected and vital roles in auxin-regulated H⁺-ATPase traffic and associated functions at the plasma membrane.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis (Arabidopsis thaliana), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs. To address this issue, we conducted an analysis of the proteins secreted by cultured wild-type, syp121, and syp122 mutant Arabidopsis seedlings. Here, we report that a number of cargo proteins were associated differentially with traffic mediated by SYP121 and SYP122. The data also indicated important overlaps between the SNAREs. Therefore, we conclude that the two Qa-SNAREs mediate distinct but complementary secretory pathways during vegetative plant growth.